Neisseria meningitidis, a commensal organism that is found frequently in the throat of healthy adolescents and which can invade the bloodstream and cause meningitis or rapidly fatal sepsis, is strictly a human pathogen. Reliable animal models of meningococcal disease have been difficult to develop. Many encapsulated strains of N. meningitidis that are highly pathogenic in humans are readily cleared from the bloodstream of commonly used experimental animals such as rabbits, mice and rats. Clearance may result in part from the role of the bacterial protein fH binding protein (fHbp), which binds human complement factor H (fH), a molecule that down-regulates complement activation. Binding of human fH to the bacterium increases resistance of the organism to complement-mediated bacterial killing and may be an important mechanism that enables N. meningitidis to circumvent innate host defenses. With N. gonorrhoeae, binding of fH is restricted to human fH, which may in part explain species-specific restriction of natural gonococcal infection (Ngampasutadol, J. et al. (2008) J Immunol 180:3426-3435).
Considerable data indicate that serum complement-mediated bactericidal antibody confers protection against meningococcal disease (Goldschneider, I. et al. (1969) J Exp Med 129:1307-1326; Goldschneider et al. (1969) J Exp Med 129:1327-1348; Borrow, R. et al. (2005) Vaccine 23:2222-2227; Balmer, P. et al. (2004) Expert Rev Vaccines 3:77-87; Andrews, N et al. (2003) Clin Diagn Lab Immunol 10:780-786; Borrow, R. et al. (2001) Infect Immun 69:1568-1573). For example, in a seminal study Goldscheider et al. investigated the ability of serum complement-mediated bactericidal antibodies to confer protection against epidemic group C meningococcal disease in military recruits (Goldschneider et al. 1969, J Exp Med 129(6): 1307-26; Goldschneider et al. 1969, J Exp Med 129(6): 1327-48). Eighty-two percent of the more than 14,000 subjects had serum bactericidal titers of ≧1:4 against the epidemic strain but 51 of the 53 subjects who subsequently developed meningococcal disease had serum bactericidal titers <1:4. The authors concluded that a titer of ≧1:4 conferred protection against disease but also pointed out that many of the estimated 2600 recruits with titers <1:4 were likely exposed to the epidemic strain and did not develop disease. Indeed more than half of a small group of recruits with SBA titers <1:4 who were demonstrated to have acquired throat carriage of the group C epidemic strain did not develop disease. Therefore, while titers ≧1:4 conferred protection, titers <1:4 were not necessarily an indicator of susceptibility to disease.
A more recent study in the United Kingdom reported that the incidence of group B meningococcal disease declined between ages 1 and 10 years without a corresponding increase in the prevalence of group B serum bactericidal titers of ≧1:4 (Trotter et al. (2007) Clin Vaccine Immunol 14(7): 863-8). This study also found a lower prevalence of serum bactericidal activity titers in young adults (about 50%) than that reported in the Goldschneider study (about 80% to 90%). Other recent studies in North America or other countries in Europe confirm that SBA titers of ≧1:4 in adults are relatively uncommon (depending on the strain, typically 10 to 25% of adults) (Mitchell et al. 1996, J Infect Dis 173(4): 1009-13; Jones et al. 2000, J Infect Dis 181(3): 1172-5; Welsch and Granoff, 2004, Infect Immun 72(10): 5903-9; Amir et al. 2005, Vaccine 23(8): 977-83; Granoff et al. 2005, Pediatr Infect Dis J 24(2): 132-136). Thus, while SBA titers of ≧1:4 were prevalent in the 1960s, they are much less common now without a corresponding increase in the incidence of meningococcal disease (rates of disease in the U.S. since 2000 are the lowest in the last 50 years). Collectively, these seroepidemiologic data are inconsistent with the hypothesis that serum bactericidal titers of ≧1:4 are required for protection against meningococcal disease. Alternative hypotheses include protection by complement-mediated bactericidal antibodies present at serum dilutions <1:4, and/or the ability of opsonic activity to confer protection in the absence of SBA.
Several standardized protocols for group A and C bactericidal assays were developed that use infant rabbit serum as a complement source instead of human serum (Maslanka, S. E. et al. (1997) Clin Diagn Lab Immunol 4:156-167; Jodar, L. et al. (2000) Biologicals 28:193-197). Although rabbit complement was selected for these protocols because of greater ease of standardization, it has been known for many years that rabbit complement augments serum bactericidal titers as compared with titers measured with human complement (Zollinger, W. D. et al. (1983) Infect Immun 40:257-264; Santos, G. F. et al. (2001) Clin Diagn Lab Immunol 8:616-623). While serum bactericidal titers measured with rabbit complement have been correlated with the effectiveness of meningococcal vaccination introduced to large populations (Borrow, R. et al. (2005) Vaccine 23:2222-2227; Balmer, P. et al. (2004) Expert Rev Vaccines 3:77-87; Andrews and Borrow (2003) Clin Diagn Lab Immunol 10:780-786), many of these sera would lack bactericidal activity if tested with human complement. Thus, the correlations observed with rabbit complement may not accurately or totally reflect the actual mechanisms by which the vaccine-induced antibodies conferred protection.
A whole blood bactericidal assay (WBA) was used to measure naturally-acquired immunity (Ison et al. (2003) Pediatr Infect Dis J 22(10): 868-73; Ison et al. (1995) Microb Pathog 18(2): 97-107), or antibody responses of children (Ison et al. (1999) Microb Pathog 27(4): 207-14) and adults (Findlow et al. (2006) Infect Immun 74(8): 4557-65) to meningococcal vaccination. This assay used fresh blood samples obtained before and after immunization from each immunized subject. As pointed out by Findlow et al (2006), WBA is not practical for measurement of responses to vaccines since fresh blood is required of each assay, and the assay described cannot be performed on stored blood or serum samples. Thus, use of fresh whole blood samples from clinical trial subjects or from patients to assess bactericidal antibodies raised in response to a vaccine is impractical or impossible. For example, comparison of pre-immune bactericidal antibodies in fresh whole blood of a subject and post-immune bactericidal antibodies in fresh whole blood of the same subject and under the same assay conditions is simply not possible—the pre-immune blood is no longer “fresh” by the time the post-immune sample is available. Further, when independent assays are performed on two samples on different days, there is less precision in determining whether the respective titers are different than when the samples are assayed simultaneously.
Opsonic activity has also been used to assess protective antibodies to N. meningitidis that might be undetected by serum bactericidal activity assays (Ross et al. 1987, J Infect Dis 155(6): 1266-75; Halstensen et al. 1991, NIPH Ann 14(2): 157-65; discussion 166-7; Lehmann et al. 1991, Apmis 99(8): 769-72; Guttormsen et al. 1993, J Infect Dis 167(6): 1314-9; Aase et al. 1995, Infect Immun 63(9): 3531-6; Aase et al. 1998, Scand J Immunol 47(4): 388-96; Lehmann et al. 1999, Infect Immun 67(12): 6526-32; Naess et al. 1999, Vaccine 17(7-8): 754-64; Quakyi et al. 1999, J Infect Dis 180(3): 747-54; Rosenqvist et al. 1999, Infect Immun 67(3): 1267-76; Plested et al. 2001, Infect Immun 69(5): 3203-13; Martinez et al. 2002, Clin Diagn Lab Immunol 9(2): 485-8; Borrow et al. 2006, Vaccine 24(24): 5093-107; Romero-Steiner et al. 2006, Clin Vaccine Immunol 13(2): 165-9; Plested et al. (2008) Clin Vaccine Immunol 15(5): 799-804). This assay typically assays test sera that has been heated to remove internal complement activity, to which exogenous rabbit or human serum is added as a complement source, and fractionated or unfractionated peripheral blood mononuclear leukocytes or a monocytic cell line grown in tissue culture are added as the phagocytic effector cells. Because paired pre- and post-immunization sera from an individual, or groups of sera from persons given different vaccines, can be assayed together in one OPA assay, the ability to determine changes in titer after vaccination, or differences in vaccine response between groups, is greater with the OPA than with the WBA described above. However, OPA assays are typically done in reaction mixtures containing diluted non-immune serum as a complement source (typically 5%, 10% or 20%). The results therefore may be less sensitive for detecting OPA bactericidal activity than with the WBA.